Project Summary/Abstract Tumor-draining lymph nodes (LN) are the first site of metastasis in most types of cancer. The extent of metastasis in the LN is often used in staging cancer progression. Notably, in recent work the applicants described novel nanoscale TRAIL-coated liposomes that when conjugated to human natural killer (NK) cells enhance their endogenous therapeutic potential in killing cancer cells both in vitro and in vivo. In this proof-of-concept study, the applicants will target these liposomes to the LN by conjugating them to NK cells, and will investigate their ability to prevent the lymphatic spread of colon cancer tumors in mice. It will be shown that targeting NK cells with TRAIL liposomes can enhance liposome retention time within regional lymph nodes to induce apoptosis in cancer cells. If successful, the proposed approach could be used to kill cancer cells within the tumor draining LN to prevent the lymphatic spread of cancer. The proposed work is organized into three Specific Aims. Specific Aim 1: To examine the mechanism of TRAIL/Anti-NK1.1 liposome therapy and test their efficacy against drug- resistant colon carcinoma in a subcutaneous LN metastasis model. Sub-aim 1.1: Examine the roles of different natural killer cell receptors on super NK cytotoxicity. Sub-aim 1.2: To test the efficacy of TRAIL/Anti-NK1.1 liposomes to treat oxaliplatin-resistant colon cancer. Oxaliplatin is a clinically important platinum-based drug however long-term treatments with oxaliplatin have been shown to lead to the acquisition of drug resistance in colorectal cancer cells. Specific Aim 2: To characterize the biodistribution, pharmacokinetics and toxicity of TRAIL/Anti-NK1.1 liposomes introduced intraperitoneally. Intraperitoneal route of liposome injection will be examined to enable efficacy studies in the orthotopic colon cancer model of Aim 3. Sub-aim 2.1: To examine the whole body biodistribution and LN pharmacokinetics of TRAIL/Anti-NK1.1 liposomes, with special focus on the mesenteric lymph nodes. Sub-aim 2.2: To assess for toxicity in response to repeated intraperitoneal injections of TRAIL/Anti-NK1.1 liposomes. Specific Aim 3: To evaluate TRAIL/Anti-NK1.1 liposome efficacy in an orthotopic model of colon cancer metastasis to the mesenteric lymph nodes and spleen-to-liver metastasis. Sub- aim 3.1: Characterize the efficacy of TRAIL/Anti-NK1.1 liposomes to treat orthotopic colon cancer metastasis to the mesenteric lymph nodes. Sub-aim 3.2:Treatment of secondary metastasis from the spleen to the liver with intravenous TRAIL/Anti-NK1.1 liposomes. Colon carcinoma cells will be injected into the spleen, a lymphatic organ, to examine whether intravenous TRAIL/Anti-NK1.1 liposome treatment can also prevent or reduce secondary metastasis from the spleen to the liver. IMPACT: This innovative TRAIL-liposome based intervention will demonstrate that NK cells can be used to eliminate tumorigenic cells in the tumor-draining LN, and thus prevent the formation of LN metastases, a currently unmet need. The success of this project will establish a new platform technology for the cellular-based delivery of receptor-ligand therapeutics for the treatment of various cancers and other diseases.